Influenza virus is a polymorphic RNA virus of Orthomyxoviridae with a diameter of about one ten thousandth millimeters (100 nm). When this virus infected human, symptoms are observed such as fever of more than 38° C., headache, arthralgia, muscular pain, and also pharyngeal pain, snivel, cough, etc. Influenza virus may adsorb to sialic acid in epidermal cells present within the nasal cavity or on the surface of the pharyngeal mucosa and be taken into cells by endocytosis, followed by release of ribonucleoprotein (RNP) into cells through membrane fusion. RNP is then transferred to the cellular nucleus where each component of the virus is synthesized using the cellular transcription/translation system and viral particles are then formed on the cellular surface using the lipid membrane from the cell. The viral particles are cut off from the cellular surface by the action of viral neuraminidase and released out of the cell.
Influenza virus is divided into three types A, B and C, among which types A and B spread epidemically. On the surface of the types A and B influenza viral particles are glycoproteins, i.e. hemagglutinin (HA) and neuraminidase (NA), which are a target antigen for protective immunization of infection. In case of type A influenza virus, in particular, there exist antigenically different subtypes, i.e. 15 subtypes for HA and 9 subtypes for NA, and viruses with various combinations of these subtypes spread widely not only in human but also in other hosts such as pig or chicken. Therefore, viruses of animal-derived subtype may invade human as zoonosis.
For influenza virus, the virus acquires infectivity through cleavage of hemagglutinin (HA) on the surface of the viral particles into HA1+HA2 and infection proceeds in multiple steps. This HA cleavage occurs through the action of a trypsin-like protease secreted from the target organs, the intestine or the airway, in case of chicken. Thus, infection in the organs may proceed through the HA cleavage and activation to exhibit pathogenicity. Also, Kido et al., from the study of mechanism of influenza viral infection, showed that cleavage and activation of the HA antigen on the viral surface by a protease (tryptase Clara) derived from the host is important for the establishment of infection (see, for instance, Non-patent reference 1).
Current influenza vaccines are either a split vaccine, which is prepared by inoculating influenza virus for the preparation of a vaccine into the allantoic cavity of growing chicken eggs for culture and propagation of the virus, concentrating and purifying the virus from the chorio-allantoic liquid by centrifugation, and treating the viral particles with ether or a surfactant to remove most of lipid components thought to be a cause of adverse side effects so as to obtain a suspension of HA fractions, followed by inactivation (removal of pathogenicity) with formalin; or a subunit vaccine prepared by disintegration of viral particles with a surfactant and further purification. Thus, since influenza vaccine is made from a fertilized egg and hence hasty mass production is not possible, its expected amount of production is prudently determined taking into consideration various conditions every year.
For a method of propagation of virus by tissue culture, a method is reported wherein human influenza viruses are cultured in Vero cells in the presence of trypsin in the culture medium at a minimum concentration of about 0.05 μg/ml (preferably about 0.05 μg/ml and about 0.5 μg/ml) throughout the viral growth cycle (see, for instance, Patent reference 1). However, this method is one for effectively isolating influenza virus from animals infected with said virus and the reference does not disclose a method suitable for the preparation of a vaccine.
Patent reference 1: Japanese patent publication No. 506081/1999
Non-patent reference 1: FEBS LETTERS, 322, p 115-119 (1993)